Hepatitis c receptor protein cd81

ABSTRACT

The present invention relates to the use of CD81 protein and polynucleic acid in the therapy and diagnosis of hepatitis C and pharmaceutical compositions, animal models and diagnostic kits for such purposes.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/231,370, filed Sep. 2, 2008, which is a continuation of U.S. patentapplication Ser. No. 11/495,151, filed Jul. 28, 2006, now abandoned,which is a continuation of U.S. patent application Ser. No. 10/859,700,filed Jun. 3, 2004, now U.S. Pat. No. 7,097,987, which is a continuationof U.S. patent application Ser. No. 09/509,612, filed Mar. 29, 2000, nowabandoned, which is a §371 application of PCT/IB98/01628, filed Oct. 6,1998, which claims priority to GB9721182.5, filed Oct. 6, 1997, andGB9813560, filed Jun. 23, 1998, from which applications priority isclaimed pursuant to the provisions of 35 U.S.C. §119 and §120, and whichapplications are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the use of CD81 protein and nucleicacid encoding this protein in the therapy and diagnosis of hepatitis Cand to pharmaceutical compositions, animal models and diagnostic kitsfor such uses.

BRIEF DESCRIPTION OF THE PRIOR ART

All publications, manuals, patents, and patent applications cited hereinare incorporated in full by reference. HCV (previously known as Non-ANon-B hepatitis—NANBV) is a positive sense RNA virus of about 10000nucleotides with a single open reading frame encoding a polyprotein ofabout 3000 amino acids. Although the structure of the virus has beenelucidated by recombinant DNA techniques (European patent applicationEPA-0318216 and European patent application EP-A-0388232), the virusitself has not been isolated and the functions of the various viralproteins produced by proteolysis of the polyprotein have only beeninferred by analogy with other similar viruses of similar genomicorganization (Choo et al PNAS USA (1991) δ 2451-2455).

The viral proteins are all available in recombinant form, expressed in avariety of cells and cell types. including yeast. bacteria, insect,plant and mammalian cells (Chien, D. Y. et al PNAS USA (1992) 8910011-10015 and Spaete. R. R. et al Virology (1992) 188 819-830). Twoproteins, named E1 and E2 (corresponding to amino acids 192-383 and384-750 of the HCV polyprotein, respectively) have been suggested to beexternal proteins of the viral envelope which are responsible for thebinding of virus to target cells.

HCV research is hindered very considerably by the limited host range ofthe virus. The only reliable animal model for HCV infection is thechimpanzee and HCV does not readily propagate in tissue culture.

In our copending International patent application PCT/IB95/00692 (WO96/05513), we describe a method employing flow cytometry to identifycells carrying the HCV receptor. We have shown that by labelling cellswith recombinant E2 envelope protein, it is possible to sort cells usingflow cytometry, isolating those cells capable of specific binding to theE2 and therefore potentially carrying an HCV receptor.

In our copending International patent application PCT/IB96/00943 (WO97/09349), we have identified a protein capable of binding to the E2envelope protein of HCV.

We have now succeeded with some difficulty in cloning the DNA encodingthe HCV receptor and have discovered, surprisingly that the DNA encodesa cellular protein known as CD81. We are not aware of any association inthe literature between CD81 and the HCV. CD81 was first identified bymonoclonal antibodies as the target of an antiproliferative antibody(TAPA-1) which inhibited in vitro cellular proliferation. Armed withthis new information and given the sequence knowledge of CD81 in thepublic databases it is now possible to design and produce an armoury oftherapeutic and diagnostic reagents against HCV.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a CD81 protein, orfunctional equivalent thereof, for use in the therapy or diagnosis ofhepatitis C(HCV). According to a further aspect of the present inventionthere is provided a compound that binds specifically to the CD81 proteinfor use in the therapy or diagnosis of HCV.

The term “CD81 protein, or a functional equivalent thereof” as usedherein means the human CD81 protein as defined by the protein sequencelisted in the SWISSPROT database (Accession No. P18582) or theEMBL/GENBANK database (Accession No. M33690) or a functional equivalentthereof. A functional equivalent of CD81 is a compound which is capableof binding to HCV, preferably to the E2 protein of HCV. Preferably, thefunctional equivalent is a peptide or protein. The term “functionalequivalent” includes an analogue of CD81, a fragment of CD81, and CD81mutants and muteins.

One region of the human CD81 protein that is shown herein to be involvedin binding to the E2 protein of HCV is the “EC2” region comprising aminoacids 113-201 of the full length human sequence shown in FIG. 1. Theinvention encompasses proteins and protein fragments containing thisregion of human CD81, or containing functional equivalents of thisregion, such as, for example, the Chimpanzee sequence identified inFIG. 1. Preferably, the functional equivalent is at least 80% homologousto the human CD81 sequence across the EC2 region of the protein,preferably at least 90% homologous as assessed by any conventionalanalysis algorithm such as for example, the Pileup sequence analysissoftware (Program Manual for the Wisconsin Package, 1996).

The term “a functionally equivalent fragment” as used herein also meansany fragment or assembly of fragments of the complete protein that bindsto HCV, preferably that binds to the E2 protein of HCV. The completeprotein may be truncated at one or both ends or domains may be removedinternally provided that the protein retains the defined function. Forexample, one or more regions of the protein responsible for membranebinding (TM1 to TM4 in FIG. 1) may be removed to render the proteinsoluble when produced by a recombinant process. The fragment of choicecomprises the extracellular loop 2 (EC2 in FIG. 1) of the CD81 protein(amino acids 113-201).

If proteinaceous, functionally equivalent fragments or analogues maybelong to the same protein family as the human CD81 protein identifiedherein. By “protein family” is meant a group of proteins that share acommon function and exhibit common sequence homology. By sequencehomology is meant that the protein sequences are related by divergencefrom a common ancestor, such as is the case between the human and thechimpanzee. Chimpanzee CD81 is thus an example of a functionallyequivalent protein that binds to HCV.

Preferably, the homology between functionally equivalent proteinsequences is at least 25% across the whole of amino acid sequence of thecomplete protein or of the complete EC2 fragment (amino acids 113-201).More preferably, the homology is at least 50%, even more preferably 75%across the whole of amino acid sequence of the protein or proteinfragment. Most preferably, homology is greater than 80% across the wholeof the sequence.

The term “a functionally equivalent analogue” is used to describe thosecompounds that possess an analogous function to an activity of the CD81protein and may, for example comprise a peptide, cyclic peptide,polypeptide, antibody or antibody fragment. These compounds may beproteins, or may be synthetic agents designed so as to mimic certainstructures or epitopes on the inhibitor protein. Preferably, thecompound is an antibody or antibody fragment.

The term “functionally equivalent analogue” also includes any analogueof CD81 obtained by altering the amino acid sequence, for example by oneor more amino acid deletions, substitutions or additions such that theprotein analogue retains the ability to bind to HCV, preferably the E2protein of HCV. Amino acid substitutions may be made, for example, bypoint mutation of the DNA encoding the amino acid sequence.

The functional equivalent of CD81 may be an analogue of a fragment ofCD81. The CD81 or functional equivalent may be chemically modified,provided it retains its ability to bind to HCV, preferably the E2protein of HCV.

It is envisaged that such molecules will be extremely useful inpreventative therapy of HCV infection, because these molecules will bindspecifically to the virus and will thus prevent internalisation of thevirus into cells. As used herein, “binding specifically” means that thefunctionally equivalent analogue has high affinity for the E2 protein ofthe HCV virus and does not bind to any other protein with similar highaffinity. Specific binding may be measured by a number of techniquessuch as Western blotting, FACS analysis, or immunoprecipitation.Preferably, the functionally equivalent analogue binds to the E2 proteinwith an affinity of at least 10⁻⁸, preferably at least 10⁻⁹ and mostpreferably greater than 10⁻¹⁰.

According to a further embodiment of the invention there is provided acompound that binds to CD81, such as a monoclonal or polyclonal antibodyto CD81, for use in the diagnosis or therapy of HCV. Preferably thecompound binds specifically to CD81 with an affinity of at least 10⁻⁸,preferably at least 10⁻⁹ and most preferably greater than 10⁻¹⁰. Suchcompounds may be used to prevent the virus binding to patient cells andbeing internalised.

The CD81 molecule is present on a number of different cell types.Ideally, the compound that binds to CD81 therefore only interacts withCD81 in the presence of HCV, so that the usual function of CD81 is notcompromised on healthy cells. Antibodies and suitable methods ofscreening for such antibodies are described in co-pending applicationsEP 96928648.3 and EP 95927918.3.

The CD81 protein, or functional equivalent thereof may be produced byany suitable means, as will be apparent to those of skill in the art. Inorder to produce sufficient amounts of CD81 protein, or functionalequivalents thereof for use in accordance with the present invention,expression may conveniently be achieved by culturing under appropriateconditions recombinant host cells containing the CD81 protein, orfunctional equivalent thereof.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known.

Two preferred methods of construction of carrier proteins according tothe invention are direct chemical synthesis and by production ofrecombinant protein. Preferably; the CD81 protein is produced byrecombinant means, by expression from an encoding nucleic acid molecule.Recombinant expression has the advantage that the production of theprotein is inexpensive, safe, facile and does not involve the use oftoxic compounds that may require subsequent removal.

When expressed in recombinant form, the CD81 protein or functionalequivalent thereof is preferably generated by expression from anencoding nucleic acid in a host cell. Any host cell may be used,depending upon the individual requirements of a particular system.Suitable host cells include bacteria, mammalian cells, plant cells,yeast and baculovirus systems. Mammalian cell lines available in the artfor expression of a heterologous polypeptide include Chinese hamsterovary cells. HeLa cells, baby hamster kidney cells and many others.Preferably, bacterial hosts are used for the production of recombinantprotein, due to the ease with which bacteria may be manipulated andgrown. A common, preferred bacterial host is E. coli.

Preferably, if produced recombinantly, the CD81 protein or functionalequivalent is expressed from a plasmid that contains a synthetic nucleicacid insert. The insertion site in the expression plasmid into which thenucleic acid encoding the CD81 protein or functional equivalent iscloned may allow linkage of the protein to a tag, such as the “flag”peptide or polyhistidine. This arrangement facilitates the subsequentpurification of recombinant protein.

According to a further aspect of the present invention, there is alsoprovided a nucleic acid molecule encoding the CD81 protein or functionalequivalent thereof, for use in the therapy or diagnosis of HCVinfection. Preferably, the nucleic acid encodes human CD81 protein. Aswill be apparent to one of skill in the art, such a nucleic acidmolecule will be designed using the genetic code so as to encode theprotein or peptide that is desired. A nucleic acid molecule according tothis aspect of the present invention may comprise DNA, RNA or cDNA andmay additionally comprise nucleotide analogues in the coding sequence.Preferably, the nucleic acid molecule will comprise DNA.

Nucleotide sequences included within the scope of this embodiment of theinvention are those hybridising to nucleic acid encoding the CD81protein under standard conditions. As used herein, standard conditionsincludes both non-stringent standard hybridisation conditions (6×ssc/50%formamide at room temperature) with washing under conditions of lowstringency (2×SSC/50% formamide at room temperature, or 2×ssc, 42° C.)or at standard conditions of higher stringency, e.g. 2×ssc, 65° C.(where ssc=0.15M NaCl, 0.015M sodium citrate, pH 7.2). Preferably theterm standard conditions refers to conditions of high stringency.

Preferably, such nucleic acid molecules will retain the ability tohybridise specifically to nucleic acid encoding CD81 or a fragmentthereof and will include nucleic acid sequences with 40% homology acrossthe whole of the human CD81 gene sequence as defined by the Pileupcommand of the GCG Program manual for the Wisconsin Package (version 9,1996). More preferably, the homology is at least 65% across the whole ofthe gene sequence. Most preferably, homology is greater than 70% acrossthe whole of the gene sequence.

Nucleic acid encoding the CD81 protein or functional equivalent may becloned under the control of an inducible promoter, so allowing preciseregulation of protein expression. Suitable inducible systems will bewell known to those of skill in the art.

Suitable vectors for the expression of the CD81 protein or functionalequivalent may be selected from commercial sources or constructed inorder to suit a particular expression system. Such vectors will containappropriate regulatory sequences, such as promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences and markergenes. Vectors may be plasmids, or viral-based. For further details seeMolecular Cloning: a laboratory manual (Sambrook et al., 1989). Manyknown techniques and protocols for the manipulation of nucleic acids andanalysis of proteins are described in detail in “Short protocols inmolecular biology”, second addition, Ausubel et al. (John Wiley & Sons1992).

Methods for the isolation and purification of recombinant proteins willbe well known to those of skill in the art and are summarised, forexample in Sambrook et al (1989). Particularly in bacteria such as E.coli, the recombinant protein will form inclusion bodies within thebacterial cell, thus facilitating its preparation. If produced ininclusion bodies, the carrier protein may need to be refolded to itsnatural conformation.

Additionally, in order to tailor precisely the exact properties of theCD81 protein or functional equivalent thereof, the skilled artisan willappreciate that changes may be made at the nucleotide level from knownCD81 sequences, by addition, substitution, deletion or insertion of oneor more nucleotides. Site-directed mutagenesis (SDM) is the method ofpreference used to generate mutated proteins according to the presentinvention. There are many techniques of SDM now known to the person ofskill in the art, including oligonucleotide-directed mutagenesis usingPCR as set out, for example by Sambrook et al., (1989) or usingcommercially available kits.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Short Protocols in MolecularBiology, Second Edition. Ausubel et al. eds., John Wiley & Sons, 1992.The disclosures of Sambrook et al. and Ausubel et al. are incorporatedherein by reference.

According to a further aspect of the invention, there is provided amethod for treating an infection of HCV comprising administering to apatient a therapeutically effective amount of CD81 protein, or afunctional equivalent thereof effective to reduce the infectivity of thevirus.

Since the infection mechanism of HCV appears to depend, in part, uponthe availability of a cell surface receptor, making available a solubleform of the CD81 protein, or a functional equivalent thereof will act asan antagonist of binding of HCV to the cellular receptor thus reducingor preventing the infection process and thereby treating the disease.

A suitable soluble form of the CD81 protein, or a functional equivalentthereof might comprise, for example, a truncated form of the proteihfrom which one or more of the transmembrane domain or domains TM1-TM4have been removed either by a protein cleavage step or, by design, in achemical or recombinant DNA synthesis. The preferred soluble form of theprotein comprises the EC2 domain (residues 113-201 as identified in FIG.1). The EC1 domain may act to increase the affinity or specificity ofthe protein for HCV.

Alternatively, a hybrid particle comprising at least oneparticle-forming protein, such as hepatitis B surface antigen or aparticle-forming fragment thereof, in combination with the CD81 proteinor functional equivalent thereof could be used as an antagonist ofbinding of HCV to the cellular receptor.

According to a still further aspect of the invention, there is provideda method for treating an infection of HCV comprising administering to apatient a therapeutically effective amount of a compound thatspecifically binds to CD81 protein, such as a monoclonal antibodydirected to CD81. The rationale behind this therapeutic strategy is thatthe binding of the cell surface receptor to another compound willprevent the binding of HCV to the receptor, so preventing the infectionprocess and thereby treating the disease.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a CD81 protein or functionalequivalent thereof, optionally as a pharmaceutically acceptable salt, incombination with a pharmaceutically acceptable carrier. According to astill further aspect of the present invention there is provided apharmaceutical composition comprising a compound that binds specificallyto the CD81 protein, optionally as a pharmaceutically acceptable salt,in combination with a pharmaceutically acceptable carrier.

The pharmaceutical composition may be in any appropriate form foradministration including oral, parenteral, transdermal andtranscutaneous compositions. The composition may be administered aloneor in combination with other treatments, either simultaneously orsequentially dependent upon the condition to be treated.

A process is also provided for making the pharmaceutical composition, inwhich a protein of the present invention is brought into associationwith a pharmaceutically acceptable carrier.

According to a further aspect of the invention, there is provided a CD81protein or functional equivalent thereof, or a compound that bindsspecifically to the CD81 protein for use as a pharmaceutical.

According to a further aspect of the invention, there is provided theuse of a CD81 protein or functional equivalent thereof or compound thatbinds specifically to the CD81 protein in the manufacture of amedicament for the treatment of an HCV infection.

The ability of a CD81 protein or functional equivalent thereof to bindto HCV permits the use of the protein as a diagnostic for HCV infection,for example in an ELISA (Enzyme linked immunosorbent assay) or RIA(Radioimmunoassay).

A soluble form of the protein could, for example, be used in an ELISAform of assay to measure neutralising antibodies in serum. Morepreferably, antibodies to CD81 will be suitable for use in this context,since these molecules will be anti-idiotypic antibodies for HCV itself.

According to a further aspect of the invention, there is provided anassay for HCV antibodies in a serum sample comprising the step ofallowing competitive binding between antibodies in the sample and aknown amount of an HCV protein for binding to a CD81 protein orfunctional equivalent thereof and measuring the amount of the known HCVprotein bound.

Preferably, the CD81 protein or functional equivalent thereof isimmobilised on a solid support and the HCV protein, which may suitablybe E2 HCV envelope protein, optionally recombinant E2 protein islabelled. The label may be a radioactive label, a peptide, an epitope,an enzyme, or any other bioactive compound. Preferably the labelcomprises an enzyme.

In an assay of this form, competitive binding between antibodies and theHCV protein for binding to the CD81 protein or functional equivalentthereof results in the bound HCV protein being a measure of antibodiesin the serum sample, most particularly, HCV neutralising antibodies inthe serum sample.

A significant advantage of the assay is that direct measurement is madeof neutralising of binding antibodies (i.e. those antibodies whichinterfere with binding of HCV envelope protein to the cellularreceptor). Such an assay, particularly in the form of an ELISA test hasconsiderable applications in the clinical environment and in routineblood screening.

Also, since the assay measures neutralising of binding antibody titre,the assay forms a ready measure of putative vaccine efficacy,neutralising of binding antibody titre being correlated with hostprotection.

In a further aspect of the invention, there is provided a diagnostic kitcomprising the CD81 protein or functional equivalent thereof. Preferablythe kit also contains at least one labelled HCV protein, optionallyenzyme labelled. The kit will also contain other components necessaryfor the analysis of the presence of HCV or anti-HCV antibodies in serum.Such components will be readily apparent to those of skill in the art.

The CD81 protein or functional equivalent thereof may be used to screenfor chemical compounds mimicking the HCV surface structure responsiblefor binding to the HCV receptor.

According to a further aspect of the invention, there is provided amethod for screening chemical compounds for ability to bind to theregion of HCV responsible for binding to a host cell, comprisingmeasuring the binding of a chemical compound to be screened to a CD81protein or functional equivalent thereof. The host cell may be anymammalian cell, preferably a human host cell.

This aspect of the invention encompasses the products of the screeningprocess whether alone, in the form of a pharmaceutically acceptablesalt, in combination with one or more other active compounds and/or incombination with one or more pharmaceutically acceptable carriers.Processes for making a pharmaceutical composition are also provided inwhich a chemical compound identified by the process of the invention isbrought into association with a pharmaceutically acceptable carrier.

The chemical compound may be an organic chemical and may contain aminoacids or amino acid analogues. Preferably however the chemical compoundis a peptide, polypeptide or a polypeptide which has been chemicallymodified to alter its specific properties, such as the affinity ofbinding to the CD81 protein or functional equivalent thereof or itsstability in vivo.

According to a further aspect of the invention, there is provided anucleic acid encoding CD81 protein or functional equivalent thereof foruse in diagnosis or therapy of HCV. The nucleic acid may encode any partof the CD81 protein, or functional equivalent thereof. Preferably, thenucleic acid encodes a portion of CD81 that binds to HCV E2. Accordingto a still further aspect of the present invention, there is provided anucleic acid encoding a peptide or polypeptide compound that bindsspecifically to CD81.

Changes to the nucleic acid may be made at the nucleotide level byaddition, substitution, deletion or insertion of one or morenucleotides, which changes may or may not be reflected at the amino acidlevel, dependent on the degeneracy of the genetic code.

The nucleic acid may be included in a vector, optionally an expressionvector permitting expression of the nucleic acid in a suitable host toproduce CD81 protein or functional equivalent thereof.

The identification of the DNA encoding the HCV receptor, namely CD81,makes available the full power of molecular biology for the molecularanalysis of HCV and in particular its infectious mechanism, offering forthe first time the possibility of designing methods of treating thevirus. PCR methods may be used to identify cells carrying the receptorand DNA molecules may be designed to act as polymerase chain reaction(PCR) primers in this connection. Although CD81 is widespread and isassociated with normal human function, the present invention includesantisense molecules inhibiting CD81 production for use in the treatmentof HCV and in the manufacture of a medicament for the treatment of HCVinfection.

The identification of polymorphisms in the CD81 protein may be found tobe associated with susceptibility to HCV infection or likely prognosis.Accordingly, the identification of the gene encoding the HCV receptorallows the evaluation of polymorphisms present throughout the humanpopulation.

According to a further aspect of the invention, there is provided anantibody to CD81 protein or functional equivalent thereof for use in thetreatment of an HCV infection and in the manufacture of a medicament forthe treatment of an HCV infection. The antibody is preferably amonoclonal antibody. Such an antibody can be used to temporarily blockthe CD81 receptor preventing infection from HCV, for example,immediately after an accidental infection with HCV-infected blood.

At present, the only available animal model of HCV infection is thechimpanzee, which is a protected species. Experiments on such animalspose a number of difficulties which together result in a veryconsiderable expense (a one year experiment with one chimpanzee can cost$70,000). Compared to this, a mouse model would be far more acceptable.Unfortunately, as described below, the HCV receptor, whilst ubiquitousin humans and found in chimpanzees, is absent in other mammals. Atransgenic mammal, for example a mouse, carrying the HCV receptor on thecell surface, perhaps expressed in greater or lesser amounts thannormally found, would be of great benefit to HCV research and thedevelopment of vaccines. Expression of mutant CD81 proteins on thesurface of cells would also be a useful research tool.

According to a further aspect of the invention, there is provided atransgenic non-human animal, suitably a mammal such as a mouse, carryinga transgene encoding a CD81 protein or functional equivalent thereof.

The transgenic animal of the invention may carry one or more othertransgenes to assist in maintaining an HCV infection.

There is also provided a process for producing a transgenic animalcomprising the step of introducing a DNA encoding a CD81 protein orfunctional equivalent thereof into the embryo of a non-human mammal,preferably a mouse. Preferably the CD81 protein or functional equivalentthereof is a human CD81 protein.

According to a further aspect of the present invention, there isprovided a CD81 protein or a functional equivalent thereof for use as aprotective immunogen in the control of HCV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sequence alignment showing the homology between human,chimpanzee, green monkey, hamster, rat and mouse CD81 gene sequences.

FIG. 1A is a schematic description of primary, secondary and tertiaryrounds of screening.

FIG. 1B is a schematic description of the final round of screening.

FIG. 2 is a FACS scan analysis of E2 bound cells.

FIG. 3 shows the dose-dependent inhibition of anti-CD81 binding to Bcells by recombinant E2. The data are expressed as % inhibition of meanfluorescence intensity.

FIG. 4 is an immunoblot showing the recognition of the membrane proteinfraction immunoprecipitated by anti-CD81 antibody. Lane 2: recombinantE2 precipitated with chimpanzee antiserum to E2; lane 3, recombinant E2precipitated with chimpanzee pre-immune serum lane 4: 20 μg of anti-CD81mAb (clone JS81 Pharmingen) precipitated with goat anti-mouse IgG, lane5: control, (20 μg of an irrelevant monoclonal antibody, anti-human CD9,ATCC) precipitated with goat anti-mouse IgG linked to protein Asepharose. Lane 1: positive control, membrane protein preparation.

FIG. 5 shows the nucleotide and deduced amino acid sequences of the EC2fragment cloned in pThio-His C and the upstream plasmid sequence codingfor the carboxyl terminus of thioredoxin and for the enterokinasecleavage site.

FIG. 6 shows the appearance of a protein band of the expected molecularmass for thioredoxin-EC2 in the extract from the induced sample.

FIG. 7 is a Coomassie Blue stained gel showing the purification ofthioredoxin-EC2.

FIG. 8 represents the nucleotide and deduced amino acid sequence of theEC2-His₆ fragment cloned into pGEX-KG as well as the upstream plasmidsequence coding for the carboxyl terminus of GST, the thrombin cleavagesite and a small glycine spacer.

FIG. 9 represents an SDS-PAGE of total proteins of the TOP10 E. coliclone which express GST-EC2-(His)₆.

FIG. 10 is a Coomassie-stained SDS-PAGE showing thrombin cleavage ofGST-EC2-(His)₆ after purification of the protein on a glutathionesepharose column.

FIG. 11 shows the dose-dependent inhibition of E2 binding tohepatocarcinoma cells by recombinant molecule expressing the majorextracellular loop (EC2) of human CD81.

FIG. 12 shows binding of HCV to CD81.

FIGS. 13-17 show the construction of nucleic acid vectors for use in thegeneration of mire transgenic for the human CD81 gene.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Recombinant E2, CellLines, Vector DNA, and Antibodies Used in the Present Study

The recombinant E2 used in this screening was produced in CHO cells(E2-CHO) (WO 97/09349). E2-CHO binds to the human T cell lymphoma cellline Molt-4. A sublime of Molt-4 (termed A2A6), was identified byexpanding individual Molt-4 cell colonies and testing for the amount ofE2-CHO that bound to the cell surface. The A2A6 subline was found tobind more E2-CHO molecule on its surface than its parental line and wastherefore chosen for the source of RNA, expecting that this subline mayhave a higher representation of the transcript encoding the E2 bindingmolecule. These cells were chosen using an assay whereby human B and Tlymphoma cells and hepatocarcinoma cell lines were incubated withrecombinant E2 expressed in mammalian cells (CHO) as described by D.Rosa et al., Proc. Natl. Acad. Sci. USA 93, 1759 (1996) and stained withbiotin-labelled anti-E2 antibodies as described by Rosa et al, (1996).Cells with the highest E2 binding ability were sorted using aFacsVantage (Becton Dickinson) and subcloned by limiting dilution.Growing clones were screened for E2 binding at the Facs and clones withthe highest Mean Fluorescence Intensity were further expanded.

WOP is a NIH3T3-derived cell which expresses polyoma T antigen (L.Dailey and C. Basilico, J. Virol. 54, 739 (1985). In this cell line,plasmids containing the polyoma replication origin can be amplifiedepisomally. Recombinant DNA constructed with pCDM8 (Invitrogen) can berecovered from selected transfectants, which contains the polyomareplication origin and is designed for the manipulation of expressionlibraries in eukaryotic cells.

A mouse monoclonal anti-E2 antibody (291A2) was used for detection ofE2-CHO bound on the cell surface of transfectants. This antibody wasobtained as follows: BALB-c mice were immunised three times withrecombinant E2 (10 μg) in complete Freund's adjuvant. Cell fusionsbetween spleen cells and non-producing myeloma cells were made accordingto standard techniques. The supernatant from fusions was then screenedfor binding to E2 bound to Molt-4 cells, so as to identify monoclonalantibodies that bound to an exposed site on the E2 molecule. The mostsuitable antibody identified in this fashion was termed 291A2.

Example 2 Construction of cDNA Library

Total RNA was extracted from the A2A6 cell line according to the methoddescribed by Chomczinsky and Sacchi (Chomczinsky, P. and Sacchi, N.(1987) Anal. Biochem. 162: 156-159). Poly(A)+ was enriched twice usingoligo(dT) cellulose. Starting from 2 μg of this RNA as a template, thedouble strand complementary DNA was synthesized using a Superscript IIcDNA synthesis kit (Life Technologies) in the presence of oligo(dT) (100ng) and random hexamer primers (100 ng). The cDNA was blunt-ended withT4 DNA polymerase, and was ligated with a BstXI linker, which allows theinsertion of the fragment into the same restriction site in thepolylinker region of the expression vector pCDM8. The linker-ligatedcDNA was phenol-extracted and ethanol precipitated using ammoniumsulphate to remove free mononucleotides, followed by Sephacryl 500chromatography (Lifetechnologies) to size-fractionate the cDNA. Thepurified cDNA fragment over 500 bp were pooled and ligated withBstXI-digested pCDM8 at a molecular ratio of approximately 1:1. Thisfinal ligation reaction was used from transformation of E. coliMC1061/P3 by electroporation using Gene-Pulser (BIORAD). A total of2×10⁶ cfu was amplified and pooled in liquid bacterial culture as a cDNAlibrary.

Example 3 Library Screening

The screening procedure was based largely on the method described byCampbell et al. (Campbell, I. G., Jones, T. A., Foulkes. W. D. andTrowsdale, J. Cancer Res. 51: 5329-5338, 1991). Enrichment was carriedout using magnetic beads (the first to the third round) (FIG. 1A) andpanning techniques (the fourth round). (FIG. 1B).

3.1 The First Round of Screening

A total of 375 μg of amplified DNA, which represents 2×10⁶ ofindependent cDNA clones. was prepared. In each transfection. 25 μg ofDNA was mixed with 10⁷ WOP cells using the Gene-Pulser electroporator(BIORAD) under the conditions of 300V/5000. Fifteen sets oftransfections were performed. After transfection, cells were incubatedat 37° C. for 2 days and then the cells were detached by trypsinizationand washed with PBS supplemented with 5% FCS and 0.5 mM EDTA twice bycentrifugation at 360×g for 10 min at 4° C. The cell pellet wasresuspended in PBS supplemented with 5% FCS and 0.5 mM EDTA (10⁷cells/ml) and then E2-CHO was added to the cell suspension at aconcentration of 10 m/ml. The cells were incubated on ice for 60 min.After washing twice with PBS supplemented with 5% FCS and 0.5 mM EDTA,the cell suspension was incubated with 291A2 antibody on ice for 30 min.After washing twice with PBS supplemented with 5% FCS and 0.5 mM EDTA,10 μl of Dynabeads (DYNAL) coupled with goat anti-mouse IG was added tothe cell suspension. The mixture was gently agitated using a CoulterMixer (Coulter) for 60 min at 4° C. Bound cells were separated usingMagnetic Particle Concentrator (DYNAL) from non-binders, according tothe manufacturer's instructions, thus enriching E2-bindingtransfectants. Plasmid DNA was recovered from the bound transfectedcells using the protocol described by Campbell et al. (Campbell, I. G.,Jones, T. A., Foulkes, W. D. and Trowsdale. J. Cancer Res. 51:5329-5338, 1991). E. coli MC1061/P3 was transformed with this plasmid byelectroporation. This DNA pool is referred to as the first enriched pool(1° EP).

3.2 The Second Round of Screening

A total of 150 μg of amplified DNA derived from 1°EP was prepared and 6sets of the transfection were performed and transfectants were enrichedusing the same condition as in the first screening. This DNA pool isreferred to as 2° EP.

3.3 The Third Round of Screening

A total of 25 μg of amplified DNA derived from 2° EP was prepared andone set of the transfection was performed. Transfectants were enrichedusing the same condition as in the first screening. During thisseparation step, transfectants formed aggregates, which might be causedby expression of irrelevant adhesion molecules. This could decrease theefficiency of enrichment because these aggregates contained magneticbeads non-specifically. To circumvent this potential problem,transfectants after the second separation by Magnetic ParticleConcentrator were diluted and plated on Terasaki plates. Approximately100 of single cells identified under microscope were pooled and plasmidDNA was extracted from them. The DNA pool prepared from this step isreferred to as 3° EP.

3.4 The Fourth Round of Screening

291A1 monoclonal antibody was incubated in a Petri dish (90 mm) at aconcentration of 10 μg/ml overnight at 4° C.

A total of 25 μg of amplified DNA derived from 3° EP was prepared andone set of transfections was performed. The transfected cells wereincubated with E2-CHO as described above, and placed onto the291A2-coated plates for 60 min at 4° C. After rinsing with a largeexcess of PBS supplemented with 5% FCS and 0.5 mM EDTA twice, the boundcells were directly treated with the lysing solution and plasmids wereextracted as described as before. This DNA pool is referred to as 4° EP.

3.4 Identification of cDNA Encoding a Molecule Binding to theRecombinant E2

DNA was isolated from single colonies derived from 4° EP. A singletransfection was performed for each plasmid preparation using the sameconditions as used for the previous screening steps, E2-binding of thetransformants was detected using a phycoerythrin-conjugated monoclonalFab fragment of goat anti-mouse Ig instead of the antibody-coupledDynabeads. Transfectants of 3° EP and 4° EP were also analyzed in thesame way. The E2-bound cells were detected on FACScan (Becton Dickinson)and analyzed with LYSIS II program (Becton Dickinson) (FIG. 2). E2-CHObinds increasingly as the purification step advances. A single clone P3showed strong E2-binding.

Example 4 DNA Sequencing Determination and Analysis

P3 contains a insert of approximately 1 kb. The DNA sequence of theinsert of the cDNA clone which confers E2-binding to WOP upontransfection was determined by an automated sequencing system using theT7 primer, whose sequence is located adjacent the cloning site of pCDM8.The sequence was screened through the GenBank databases using the GCGprograms on a UNIX computer. This analysis revealed that the 5′ part ofP3 insert is identical to human CD81 (TAPA-1). Restriction analysis ofP3 using three enzymes (BstXI), HincII and NcoI) also agreed with therestriction map of human CD81 cDNA.

Example 5 Binding of CD81 to Recombinant E2

Anti-CD81 antibodies were used to assess the interaction between E2 andCD81. EBV-B cells were incubated with increasing concentrations ofrecombinant E2 for 1 hour at 4° C. and then stained with an anti-CD81monoclonal antibody (clone JS-81, Pharmingen). As shown in FIG. 3,recombinant E2 was found to competitively inhibit the binding ofanti-CD81 antibodies to EBV transformed B-cell lines (EBV-B cells). Thedata are expressed as % inhibition of mean fluorescence intensity (Rosaet al., 1996).

In addition, E2 reacts in Western blot with anti-CD81 precipitatedmaterial (FIG. 4). This Figure shows E2 recognition of membrane proteinfraction immunoprecipitated by anti-CD81 antibody. Approximately 300 μgof membrane protein extract prepared from the A2A6 cell line weresolubilised in 8 mM CHAPS in PBS pH 7.4, incubated with 10 μgrecombinant E2 (lanes 2 and 3), with 20 μg of anti-CD81 mAb (clone JS81;Pharmingen) (lane 4), or as control, with 20 μg of an irrelevantmonoclonal antibody (anti-human CD9, ATCC) (lane 5) for 2 hours at 4°C., and finally precipitated with chimpanzee antiserum to E2 (lane 2),chimpanzee pre-immune serum (lane 3), or goat anti-mouse IgG (lanes 4and 5) bound to protein A sepharose (CL-4B, Pharmacia). The pellet wasdissolved in Laemmli buffer and subjected to SDS-PAGE under non-reducingconditions. After electroblotting, the PVDF membrane (Millipore) wasincubated overnight with 1 μg/ml of recombinant E2 at room temperature,and for 2 hours with 291A2 anti-E2 monoclonal antibody. E2 binding toimmunoprecipitated proteins was detected with an anti-mouse IgGperoxidase-conjugated polyclonal aniibody (Amersham). As a positivecontrol membrane proteins also were loaded on the gels (lane 1). Themobility of molecular weight standards is indicated on the left inkilodaltons.

CD81 is also expressed on fresh lymphocytes and hepatocytes asdemonstrated by immunohistochemical staining with biotin-labelled-E2 oranti-CD81 (data not shown).

To assess whether CD81 could mediate the internalisation of ligands. weexploited the fact that CD81 forms a complex with CD19 and CD21 on thesurface of B lymphocytes (D. T. Fearon and R. H. Carter, 1995, Annu.Rev. Immunol. 13, 127). B cells were incubated with E2 at 37° C. fordifferent times, after which CD19 or CD21 levels on the cell surfacewere measured by immunofluorescence. Incubation of B cells with E2resulted in down-regulation of both CD19 and CD21 (data not shown). Itthus seems as if CD81 is able to mediate the internalisation of boththese ligands.

Example 6 The Major Extracellular Loop of CD81 Binds Recombinant E2 andViral Particles

To map the CD81 domain that binds E2 protein our efforts were focused onthe EC2 hydrophilic extracellular loop of the protein. This fragment wasexpressed in E. coli as a Thioredoxin-EC2 fusion protein that has anenterokinase site between thioredoxin and EC2, and as a GST-EC2 fusionprotein which has a thrombin site between GST and EC2 and ahexa-histidine tag added to the carboxyl-terminus of the protein. Weshow that both proteins are expressed and are able to bind HCV E2. Incompetition experiments we also show that the purified fusion proteinsand the EC2-His fragment excised from GST-thrombin-EC2-(His)₆ are ableto inhibit the binding of E2 on the surface of CD81 expressing cells.

6.1 Cloning of EC2 in pThio-His.

FIG. 5 shows the nucleotide and the deduced amino acid sequences of theEC2 fragment cloned in pThio-His C and the upstream plasmid sequencecoding for the carboxyl terminus of thioredoxin and for the enterokinasecleavage site. As shown, EC2 is fused in frame with thioredoxin throughthe enterokinase site. which can be exploited to remove thioredoxin fromthe fusion protein.

The fragment coding for EC2 was PCR-amplified from the plasmid pCDM8/P3using the following oligodeoxynucleotides:

Forward BL                          EC25′GGCGGGGGTGGATCCGGGGGTGGAGGCTCGAGCTTTGTCAACAAGGAC C3                          XhoI Phe Val Asn Lys AspReverse BL           EC2 5′CCCCAAGCTT TCA CAG CTT CCC GGA GAA GAG GTC ATC G3′       HindIII Stop Leu Lys Gly Ser Phe Leu Asp Asp

Using standard cloning techniques (Sambrook et al., 1989) the PCRproduct was double-digested with XhoI and HindIII, ligated to pThio-HisC (Invitrogen) digested with the same restriction enzymes, andtransformed into Top 10 E. coli cells. After selection of thetransformants by restriction enzyme analysis and DNA sequencing of theplasmids, a correct construct coding for the expectedthioredoxinenterokinase site-EC2 fusion protein was identified. Glycerolbatches of selected clones were stored to −80° C.

Total protein extracts of the thioredoxin-EC2 expressing clone beforeand after IPTG addition, were subjected to SDS-PAGE to analyse proteinexpression. FIG. 6 clearly shows the appearance of a protein band of theexpected molecular mass (23.4 kDa) in the extract from the inducedsample. The figure also shows the reactivity of the fusion protein withE2. The TOP10 E. coli clone containing the pThio-hisC-EC2 plasmid and aTOP10 clone containing the pThio-His C plasmid devoid of insert wereinduced, soluble protein extracts were prepared from both clones andsubjected to Far Western Blot with E2 protein. For this blot, proteinsamples were brought to 1× loading sample buffer (LSB) (5% w/v SDS, 10%v/v glycerol, 62.5 mM Tris-HCl, 0.05% Bromophenol Blue) using a 3×LSBsolution. The samples were run onto a 15% polyacrylamide gel andtransferred to a PVDF membrane (Immobilon-P, Millipore). The membranewas incubated for 30 min in blocking solution (PBS, 10% w/v non-fatdried milk, 0.05% v/v Tween 20). Following an incubation of 15 hours at4° C. with blocking solution containing 1 μg/ml of CHO-E2, the membraneswere incubated for 2 hours with the 291A2 anti-E2 monoclonal antibodydiluted 1:250, and for 1 hour with a peroxidated goat antimouse Igantibody (Sigma) diluted 1:2000. Three washing steps between allincubation steps were performed using blocking solution, which was alsoused to dilute the antibodies. After a final wash with PBS the membraneswere incubated for 1 min with luminol (ECL, Amersham) and exposed onHyper-film (Amersham).

As can be seen from these Figures, a band corresponding to the molecularweight of Thioredoxin-EC2 was visible in the lane where the solubleproteins from the pThio-His C-EC2 were loaded. Such a band was absent inthe lane where the soluble proteins of the pThio-His C clone wereloaded.

6.2 Purification of Thioredoxin-EC2

For the purification of thioredoxin-EC2 the following procedure wasdeveloped:

1) osmotic shock of the cells, 2) protein precipitation with 30%saturation ammonium sulphate, and 3) IMAC. After osmotic shock about 50%of the fusion protein was released from the cells together withcontaminant proteins. The ammonium sulphate precipitation resulted in apellet which contained thioredoxin-EC2 devoid of the bulk of contaminantproteins. IMAC of the resuspended precipitate resulted in a fusionprotein which was about 85% pure as assessed by SDS-PAGE. With thisprocedure we purified 5 mg thioredoxin-EC2 from a litre of culture. Thisprocedure is set out in detail below.

The E. coli clone expressing Thioredoxin-EC2 was inoculated in 500 ml LBmedium containing 100 μg/ml ampicillin. At OD₆₀₀=0.5, 0.5 mM IPTG wasadded to the culture and growth was continued at 37° C. for additional3.5 hours. The culture was then centrifuged at 4000×g for 10 mM at 4°C., the cell pellet was resuspended with 50 ml ice cold hypertonicsolution (20 mM Tris-HCl, 2.5 mM EDTA, 20% sucrose, pH 8) and left onice for 10 min. The resuspended cells were centrifuged again as aboveand the pellet was resuspended in hypotonic buffer (20 mM Tris-HCl, 2.5mM EDTA, pH 8) to osmotically shock the cells. After 20 min at 0° C. thesuspension was centrifuged at 12.000×g for 10 min at 4° C., thesupernatant was brought to 30% NH₂(SO₄)₂ using a room temperaturesaturated solution of the salt. The suspension was incubated overnightat 4° C. and then centrifuged at 10.000×g for 10 min. The pellet wasresuspended using 15 ml of 20 mM Phosphate buffer, 500 mM NaCl, pH 6,clarified by centrifugation, and loaded on a 2 ml column of Nickelactivated Chelating Sepharose Fast Flow (Pharmacia) equilibrated in thesame buffer.

After adsorption, the column was washed with 10 ml of the equilibriumbuffer (flow rate 0.5 ml/min), and then the Thioredoxin-EC2 was elutedusing a 30 ml gradient 0-50 mM Imidazole in 20 mM Phosphate buffer, 500mM NaCl, pH 6 followed by an isocratic elution with 10 ml of 400 mMimidazole. 2.4 ml fractions were collected. The fractions containing therecombinant protein were pooled, dialysed against PBS, and stored to−20° C. Proteins were analysed by means of SDS-PAGE and protein contentwas assayed by the Bradford method using BSA as a protein standard.

Purified Thioredoxin-EC2 is shown in FIG. 7.

6.3 Cloning of EC2-(His)₆ in pGEX-KG

FIG. 8 represents the nucleotide and deduced amino acid sequence of theEC2-(His)₆ fragment cloned in pGEX-KG as well as the upstream plasmidsequence coding for the carboxyl terminus of GST, the thrombin cleavagesite, and a small glycine spacer. As shown, EC2 is fused in frame withGST through the thrombin site, which can be exploited to remove GST fromthe fusion protein. The glycine-rich spacer, located between thrombinsite and EC2, facilitates the cleavage of the fusion protein by thrombin(Guan, K. L. and Dixon, J. E. (1991) Anal. Biochem. 192, 262-267).

The fragment coding for EC2 was PCR-amplified from the plasmid pCDM8/P3using the following oligodeoxynucleotides:

EC2 Forward         EC2 5′ CAAAAGGAATTCTA TTT GTC AAC AAG GAC CAG ATC GCC AAG3′         EcoRI Phe Val Asn Lys Asp Gln Ile Ala LysReverse BLH His tag EC2 5′CCCCAAGCTTTCAATGATG ATG ATG ATG ATG CAG CTT CCC GGA GAAG3′      HindIII Stop His His His His His His Leu Lys Gly Ser Phe

The PCR product was digested with XhoI and HindIII, ligated to pGEX-KG(Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem. 192, 262-267)digested with the same restriction enzymes, and transformed into TOP10E. coli cells. After selection of the transformants by restrictionenzyme analysis and nucleotide sequencing of the plasmids, a plasmidhaving the expected size of the insert was found to have also thecorrect EC2-(His)₆ sequence in frame with the upstream thrombin and GSTcoding sequence. The plasmid prepared from the selected TOP10 clone wasthen transformed into BL21 cells. Glycerol batches of selected cloneswere stored to −80° C.

FIG. 9 represents an SDS-PAGE of total proteins of the TOP10 E. coliclone which expresses GST-EC2-(His)₆. This analysis clearly shows thatin the extract of the induced sample a protein band with the expectedmolecular mass (39 kDa) was present. The corresponding Far Western Blotclearly shows the E2 specifically reacts with the fusion protein.

6.4 Purification of GST-EC2-(His)₆

The GST-EC2-(His)₆ fusion protein was purified on a glutathionesepharose column and digested with thrombin (FIG. 10). After digestion,the EC2-(His)₆ moiety was further purified by two additionalchromatographic steps consisting of a glutathione sepharose column toremove the GST fragment and IMAC chromatography. This procedure isdetailed below.

A single colony of an E. coli clone expressing the GST-EC2 fusionprotein was inoculated in 10 ml LB, 100 μg/ml Amp and cells were grownovernight at 37° C. The culture was then inoculated in 500 ml of mediumand when OD₆₀₀=0.5 was reached 0.5 mM IPTG was added. After 3.5 hoursthe cells were harvested by centrifugation, resuspended with 9 ml PBSand disrupted with two passages at 18.000 psi using a French Press (SLMAminco). The lysate was centrifuged at 30.000×g and the supernatant wasloaded on a column of 1 ml of Glutathione Sepharose 413 (Pharmacia)equilibrated in PBS.

The column was washed with 10 ml PBS, and eluted with 4 ml of 50 mMTris-HCl, 10 mM reduced glutathione, pH 8. The eluted proteins weredialysed against PBS and stored to −20° C.

6.5 Digestion of GST-EC2-(His)₆ with Thrombin and Purification ofEC2-(His)₆

9.6 mg of protein recovered from the glutathione sepharose column weredigested with 22 units of thrombin (Pharmacia) for 8 hours at roomtemperature, then the enzyme was inactivated using 0.13 mM PMSF (Sigma).The reaction mixture was then dialysed against PBS and loaded into 0.5ml of GST-sepharose column equilibrated in PBS. The column was washedwith 1 ml of PBS. The flow-through and the wash were pooled and loadedinto 0.250 ml of Nickel-activated chelating sepharose column. EC2-(His)₆was recovered from the column eluting with 1 ml of 20 mM phosphatebuffer, 500 mM NaCl, 400 mM imidazole, pH 7.8. A dialysis was thenperformed against PBS.

Example 7 Binding of CD81 Fragment to Virus

The proteins containing the human, but not the mouse EC2 loop of CD81,bound to E2 in western blot (data not shown) and inhibited binding of E2to human cells (FIG. 11).

The chimeric proteins were coated on polystyrene beads and incubatedwith an infectious plasma containing known amounts of viral RNAmolecules. After washing, the bead-associated virus was assessed byquantitative RT-PCR for the amount of bound HCV RNA. This experiment wasperformed as set out below.

Polystyrene beads (¼ inch diameter) (Pierce) were coated overnight withpurified EC2 recombinant protein in citrate buffer pH4 at roomtemperature. After saturation for one hour with 2% BSA in 50 mM TrisC1pH 8, 1 mM EDTA. 100 mM NaCl (TEN) buffer, each bead was incubated at37° C. for 2 hours in 200 μl TEN-diluted infectious chimp plasmacontaining 5×10⁵ HCV RNA molecules.

For inhibition experiments, the EC2-coated polystyrene beads wereincubated with 10 μg/ml of purified monoclonal antibodies for one hourat room temperature before incubation with the virus. Each bead waswashed 5 times with 15 ml TEN buffer in an automated washer (Abbot) andviral RNA was extracted using the Viral Extraction Kit (Qiagen). RNA (8ml) was reverse-transcribed at 42° C. for 90 minutes in 20 ml Buffer A1.5 (Perkin Elmer Taq Man) containing 100 pmol of the HCV antisenseprimer CGGTTCCGCAGACCACTATG, 40 U. RNAsin (Promega), 5 nmol dNTPs, 110nmol MgCl₂, 10U M-MuRT (Boheringer). cDNA (20 ml) was amplified using aPerkin-Elmer ABI 7700 Sequence Detection System (45 cycles) in 50 mlBuffer A containing 100 pmol of the HCV sense primerTCTTCACGCAGAAAGCGTCTA, 5 pmol of the fluorescent detection probe5′(FAM)TGAGTGTCGTGCAGCCTCCAGGA(TAMRA) (kindly provided by David Slade,Pharmacia and Upjohn), 15 nmol dNTPs, MgCl₂ and 1.25U Taq Gold(Perkin-Elmer, Foster City, Calif.). All reactions were quantified usingHCV (genotype 1a) infected plasma (bDNA titer of 30 mEq/ml) to generatea standard curve. Sequence Detector Software from Perkin-Elmer has beenpreviously described (U. E. Gibson, C. A. Heid and P. M. Williams,Genome Res. 6, 995 (1996)).

As shown in FIG. 12, the molecules containing the human CD81extracellular loop bound HCV in a concentration-dependent fashion, andpre-incubation of the chimeric proteins with anti-CD81 antibodiesinhibited virus binding. Furthermore, serum from chimpanzees which wereprotected from homologous challenge by vaccination with recombinantE1/E2 envelope heterodimer (Q.-L. Choo et al. Proc. Natl. Acad. Sci. USA91, 1294 (1994)) completely inhibited HCV binding to bead-coated-CD81,while serum from vaccinated and non-protected animals did not (data notshown).

These data demonstrate that expression of human CD81, and in particularits major extracellular loop are sufficient for binding not only E2 butalso HCV particles. Given the wide distribution of CD81 (S. Levy, S. C.Todd and H. T. Maecker, Annu. Rev. Immunol. 16, 89 (1998), these resultsimply that HCV binds and may be internalised by a variety of cells otherthan hepatocytes. Indeed, HCV RNA has been found in T and B lymphocytesand monocytes (K. Blight, R. R. Lesniewski, J. T. LaBrooy and E. J.Gowans, Hepatology 20, 553 (1994); P. Bouffard et al., J. Infect. Dis.166, 1276 (1992); Zignego et al., J. Hepatol. 15, 382 (1992)). Whethervirus binding is followed by entry and infection in all cell types isnot clear because of the lack of an efficient HCV culture system invitro. It may well be that CD81 is an HCV attachment receptor and thatadditional factors are required for viral fusion or infectivity.

CD81 participates in different molecular complexes on different celltypes, a fact that may influence its capacity to serve as a receptor forHCV infection or to deliver regulatory signals to target cells. Forinstance, it associates with integrins on epithelial and hematopoieticcells (F. Berditchevski, M. Zutter and M. E. Hemler, Mol. Biol. Cell 7,193 (1996); B. A. Mannion, F. Berditchevski, S.-K. Kraeft, L. B. Chenand M. E. Hemler, J. Immunol. 157, 2039 (1996)), whereas it is part of asignaling complex containing CD21, CD19 and Leu 13 on B cells (L. E.Bradbury, G. S. Kansas, S. Levy, R. L. Evans and T. F. Tedder, J.Immunol. 149, 2841 (1991)). This complex has been shown to facilitateantigen specific stimulation by lowering the activation threshold of Bcells (D. T. Fearon and R. H. Carter, Annu. Rev. Immunol. 13, 127(1995)). It is worth noting that HCV appears to use a molecule that ispart of the same complex containing the EBV receptor (CD21) (N. R.Cooper, M. D. Moore and G. R. Nemerow, Annu. Rev. Immunol. 6, 85(1988)), and the ability of EBV to activate and immortalise Blymphocytes is well documented.

Example 8 Construction of Transgenes

The following constructs were designed and made in order to generatemice transgenic for human CD81.

1. Addition of Splicing and Polyadenylation Signals of RabbitBeta-Globin Gene to the Human CD81 cDNA Fragment.

The human CD81 cDNA fragment from the pCDM8/P3 clone was transferredinto a pBluescript KS II(+) vector (Stratagene) and was then insertedinto the pSPP plasmid (derived from BMGSC expression vector, a kind giftfrom Dr. Karasuyama, Basel Institute for Immunology) between twofragments, one containing the second intron and the other containing thepolyadenylation signal of the rabbit beta-globin gene (position 902-1547and 1543-2081, respectively, GenBank accession No. M12603) (pSR1P inFIG. 11). The resulting recombinant DNA fragment was excised from thepBluescript KSII(+) vector (Stratagene) by SalI (at 5′ end) and BamHI(at 3′ end).

2. Creation of a Transgene for Ubiquitous Expression of Human CD81

The SalI-BamHI fragment of the pSR1P insert was inserted into thecompatible restriction sites of pCAGmcs, a modified plasmid of pCAGGS (akind gift from Dr. J. Miyazaki at Osaka University, Japan, underrestricted permission), which contains chicken beta-actin promoter andhuman cytomegalovirus enhancer (Niwa, H. et al., Gene 108, p 193 (1991).(pCAGSR1Pp in FIG. 12). The 3.8 kb EcoRI-BamHI fragment was submitted tozygote injection.

3. Creation of a Transgene for Liver-Specific Expression of Human CD81

The SalI site of pSR1P was converted to a BamHI site by BamHI linkerligation after blunt-end formation with Klenow fragment of E. coli DNApolymerase I. This BamHI fragment was inserted into the BamHI site ofthe ALB e/p plasmid, carrying the mouse albumin promoter and enhancer(Pinkert, C. A. et al., Genes Dev. 1, p 268 (1987) (received from Dr. F.Chisari, Scripps Research Institute. La Jolla, San Diego). (pAIbSR1P inFIG. 13) The 4.5 kb NotI-EcoRV fragment was submitted to zygoteinjection.

4. Creation of a Transgene for B Lymphocyte-Specific Expression of HumanCD81

700 by BamHI fragment of the mouse immunoglobulin heavy chain enhancer(a kind gift from Dr. A. Kudo, Basel Institute for Immunology) and 2.3kb XbaI-SacI fragment of the mouse kappa light chain promoter wassubcloned into a pBluescript KSII(+) vector. The Sad site was convertedto a HindIII site by HindIII linker ligation described above. The BamHIsite of pCAGSR1P was first converted to Nod site. Then the promoterregion of the modified pCAGSR1P construct was removed by EcoRI-HindIIIrestriction digestion and replaced with the immunoglobulinpromoter-enhancer fragment. (pEhKpSRIP in FIG. 15) The 5.2 kbEcoRI-BamHI fragment was submitted to zygote injection.

Together, our data indicate that CD81 is an attachment receptor for HCVand may provide new insight into the mechanisms of HCV infectionpathogenesis. Since CD81 associates with an activation complex on thesurface of B cells, the present finding may explain the pathogenesis ofHCV associated cryoglobulinemia, even if there is no viral replicationin B cells. Moreover, the identification of the interaction between HCVand CD81 may help in mapping conserved neutralising epitopes on thevirus envelope which should be important to develop effective vaccinesand to provide a decoy receptor for viral neutralisation.

1. A transgenic, non-human mammal comprising a nucleotide sequenceencoding a CD81 protein.
 2. The transgenic; non-human mammal of claim 1,wherein the mammal is a mouse.
 3. The transgenic, non-human mammal ofclaim 1, wherein the nucleotide sequence is the human CD81 nucleotidesequence.
 4. The transgenic, non-human mammal of claim 2, wherein thenucleotide sequence is, the human CD81 nucleotide sequence.
 5. A methodof producing a transgenic non-human mammal comprising introducing anucleotide sequence encoding a CD81 protein into the embryo of saidnon-human mammal.
 6. The method of claim 5, wherein the non-human mammalis a mouse.
 7. The method of claim 5, wherein the nucleotide sequence isa CD81 nucleotide sequence.
 8. The method of claim 6, wherein thenucleotide sequence is a CD81 nucleotide sequence.
 9. The transgenic,non-human mammal of claim 1, wherein the mammal provides for ubiquitousexpression of CD81.
 10. The transgenic, non-human mammal of claim 1,wherein the mammal provides for liver-specific expression of CD81. 11.The transgenic, non-human mammal of claim 1, wherein the mammal providesfor B lymphocyte-specific expression of CD81.